1. 1 23
Mycoscience
ISSN 1340-3540
Volume 53
Number 5
Mycoscience (2012) 53:391-395
DOI 10.1007/s10267-011-0178-4
Additions to Gliocephalotrichum species
(anamorphic Hypocreales) from fruit litter
of the medicinal plant Terminalia chebula
in the Western Ghats, India
S. K. Singh, L. S. Yadav, P. N. Singh,
R. Sharma & G. Mukherjee

2. 1 23
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3. SHORT COMMUNICATION
Additions to Gliocephalotrichum species (anamorphic
Hypocreales) from fruit litter of the medicinal plant
Terminalia chebula in the Western Ghats, India
S. K. Singh • L. S. Yadav • P. N. Singh •
R. Sharma • G. Mukherjee
Received: 29 September 2011 / Accepted: 26 December 2011 / Published online: 24 January 2012
Ó The Mycological Society of Japan and Springer 2012
Abstract Two species of Gliocephalotrichum were iso-
lated from fallen fruits of the medicinal plant Terminalia
chebula collected from the forests of Western Ghats, India.
On the basis of morphological characters and ITS1–5.8S–
ITS2 sequence similarity, the fungi have been identified as
Gliocephalotrichum longibrachium and G. bulbilium. For
the former species, this is the first report of its occurrence
from India, whereas the latter, showing significant mor-
phological variability, has been known previously from
India.
Keywords Anamorphic fungi Á ITS sequence Á
Morphology Á New record
The Western Ghats comprise mountain ranges in south-
western India that are rich in evergreen rainforests. They
are recognized as biodiversity hotspots by virtue of their
rich flora and fauna, including endemic species. Our
studies on fungal diversity have led to the identification
of several novel fungi from this region (Singh et al.
2009, 2010; Senthilarasu et al. 2010; Karandikar and
Singh 2010; Rajeshkumar et al. 2010). Terminalia chebula
L. (Combretaceae), a medicinal tree growing extensively in
the region, is widely used in ayurvedic medicines in India
for ailments such as wounds, ulcer, inflammation, skin
diseases, jaundice, and renal calculi. From decaying fruits
associated with the moist litter (fruit litter), two fungi
referable to the genus Gliocephalotrichum Ellis & Hes-
seltine (1962) were isolated and investigated for their
taxonomic identity and in vitro cultural characteristics.
Identification of the two fungi as Gliocephalotrichum
bulbilium J.J. Ellis & C.W. Hesseltine and Gliocephalo-
trichum longibrachium Decock & S. Huret has been based
on morphological characteristics of the sporulation as well
as sequencing of internal transcribed spacer (ITS) regions
of the rDNA. One species, G. longibrachium Decock &
S. Huret, is a first report for the country (Bilgrami et al.
1991; Jamaluddin et al. 2004), whereas G. bulbilium J.J.
Ellis & C.W. Hesseltine is redocumented with taxonomic
details recorded from in vitro culture lacking in previous
reports (Jamaluddin et al. 1972; Arya 1994). The present
study describes the two Gliocephalotrichum species and
reveals their intraspecific morphological and molecular
variation.
The decaying fruits of T. chebula (Combretaceae)
collected from Mahabaleshwar forests (178550
1500
N,
73°390
2100
E) of Maharashtra, India, in October 2008 were
given a mild surface sterilization with 70% ethanol followed
by incubation in a moist chamber at 25°C. From the cultures
that developed, spores were picked up and streaked on agar
plates, and spore suspensions in sterile water were plated out
using the dilution poured plate technique. Potato dextrose
agar (PDA), V-8 juice agar (V8), cornmeal agar (CMA), and
malt extract agar (MEA) supported good growth and sporu-
lation, and cultural characters of the fungi were studied on
these media after incubation at 25°C. Specimens mounted in
lactophenol-cotton blue were photographed with an Olympus
The herbarium sample is deposited in Ajrekar Mycological
Herbarium (AMH 9279) at MACS’ Agharkar Research Institute,
Pune, India (AMH, according to Holmgren et al. 1990), and the in
vitro pure cultures isolated from designated herbarium specimen are
deposited in National Fungal Culture Collection of India (NFCCI-
WDCM 932), MACS’ Agharkar Research Institute, Pune, India.
S. K. Singh (&) Á L. S. Yadav Á P. N. Singh Á R. Sharma Á
G. Mukherjee
National Facility for Culture Collection of Fungi, Mycology and
Plant Pathology Group, MACS’ Agharkar Research Institute,
G.G. Agarkar Road, Pune 411004, India
e-mail: singhsksingh@gmail.com
123
Mycoscience (2012) 53:391–395
DOI 10.1007/s10267-011-0178-4
Author's personal copy

4. CX-41 light microscope. The sizes of different fruiting
structures recorded are the extreme values of 50 measure-
ments, and in some cases an average of these measurements is
shown in parentheses.
The specimen (dried fruit) has been deposited in Ajrekar
Mycological Herbarium (AMH, according to Holmgren
et al. 1990) with the accession no. AMH 9279; in vitro pure
culture isolated from T. chebula fruits has been deposited
in National Fungal Culture Collection of India (NFCCI-
WDCM 932) with the accession numbers NFCCI 1494 (G.
bulbilium) and NFCCI 1921 (G. longibrachium), MACS’
Agharkar Research Institute, Pune, India.
Mycelial culture was homogenized in a FastPrep 24 tissue
homogenizer (MP Biomedicals, Germany) followed by
nuclear DNA extraction by the CTAB method of Gra¨ser et al.
(1999).The polymerasechainreaction(PCR) amplificationof
the ITS region (ITS1–5.8S–ITS2) and subsequent sequencing
was performed as described earlier (Singh et al. 2010).
Ribosomal DNA sequences have been deposited in GenBank
with the accession numbers HQ171605 (G. bulbilium) and
HQ171606 (G. longibrachium).
For phylogenetic study, related sequences (including all
known Gliocephalitrichum species) were retrieved in
FASTA format from GenBank. Sequence alignment of ITS
region of 30 strains was done using CLUSTAL W and
analyzed by the neighbor-joining method using the Kimura
two-parameter model by MEGA ver. 5 (Tamura et al.
2011). ITS sequences of Gliocephalotrichum species iso-
lated from India were also aligned manually with the
authentic sequences of the types of all known species of the
genus for identifying variable loci. The sequence alignment
has been submitted in TreeBASE database.
Comparative analysis of morphological features when
cultivated on different media such as PDA, CMA, V-8
juice agar, and MEA showed wide variation in number and
position of the sterile arms, conidiophore length, and
number and position of the penicillus, particularly in
the isolate referable to G. bulbilium (Nicot 1967; Decock
et al. 2006; Singh et al. 2010). The technical descriptions
and the taxonomic identity of the two isolates are as
follows.
Gliocephalotrichum longibrachium C. Decock et S. Huret.
Mycologia 98:489, 2006 Fig. 1a–c
Colonies on PDA 75–80 mm in diameter in 5 days, floc-
cose, center pale mouse grey, and margin olivaceous
buff with abundant aerial mycelium and conidial masses.
Chlamydospores abundant. On V8, 80–85 mm in diameter
in 5 days, hyaline, later turning to yellow with abundant
sporulation. On CMA, 80–85 mm diameter in 5 days,
floccose, raised, whitish, aerial mycelium turns yellowish
to olivaceous with sporulation.
Emerging hyphae branched, hyaline to pale yellow,
5–11 lm wide. Conidiophores mononematous, macrone-
matous, arising directly from submerged hyphae and from
chlamydospores, hyaline to subhyaline,1–2-septate (rarely
1–3-septate), 140–225 lm long (avg. 203.3 lm),
7.5–15 lm wide at the base (avg. 11.56 lm), 6.5–11 lm
wide at the apex, base bulbous and swollen, conidiophores
bearing a compact penicillate head and sterile arms at the
apex; penicillus with successive branches; primary 3–6,
cylindrical, thick walled, 10–20 9 3–6.5 lm; secondary
3–4, cylindrical to slightly clavate, 6–10 9 3–5 lm; ter-
tiary (3–4), 5–8 9 2.5–4 lm, bearing 2–3 quaternary
branches or 3–6 phialides with conidia. Conidia hyaline,
cylindrical to fuisoid, base rounded, without globules,
5–8.5 9 1–2.0 lm. Sterile arms 2–7, subtending to the
main penicillus, arising at right angle from conidiophores,
growing upward above the conidial heads, hyaline, septate,
thick walled, apical cell spatulate to clavate, 50–250 lm
long, 3.5–7.5 lm wide at the base, 2.5–3.5 lm at the apex.
Chlamydospores rounded in long chain, or in irregularly
shaped clusters, thick walled, light brown to brown, indi-
vidual cells 6–28 9 8–30 lm.
Material examined: India, Maharashtra, Mahabalesh-
war; on decomposing fruits of Terminalia chebula, August
2009, L.S. Yadav, in vitro culture no. NFCCI 1921.
Gliocephalotrichum bulbilium Ellis J.J. et C.W. Hessel-
tine. Bull. Torrey Bot. Club 89: 22, 1962 Fig. 1d–f
Colonies on PDA 80 mm in 3 days at 25°C, floccose, center
buff, margindull white tobuff, reverse dull white, sporulating
well with chlamydospores at center. Conidiophores mo-
nonematous, macronematous, arising from submerged
hyphae, hyaline to subhyaline, minutely roughened, 4–12
septate, 187–575 9 7.5–15 lm (avg. 332 9 10.6), bearing
a compact penicillus-like head. Penicillus hyaline with
3–5 successive branches; primary branches 15–35 9
7.5–12.5 lm (avg. 27.9 9 8.08), secondary branches (2–4),
12.5–15 9 5–12.5 lm (avg. 13.3 9 6.1); tertiary branches
(2–3), 7.5–8.0 9 4.5–5.0 lm (avg. 7.7 9 4.6). Conidia
variable in shape, oblong to cylindrical or allantoid, straight
to curved, sometimes with curved tip, hyaline, 6–12.5 9
2–2.5 lm (avg. 9.8 9 2.2 lm). Sterile arms (2–9) directly
subtending to the penicillus, arising at right angle from con-
idiophores, sometimes arising below at irregular distance
from penicillus and from two different positions at conidio-
phores,hyaline,thickwalled,5–12septate,350–550 lmlong
(avg. 418), base 5–7.5 lm wide, middle 2.5–3.5 lm, and
apex 1–2 lm. Chlamydospores intercalary, muriform, glo-
bose to elongated, thick walled, light, normal to golden
brown, 50–70 9 50–85 lm diameter (avg. 50 9 69.2).
Material examined: India, Maharashtra, Mahabalesh-
war; on decomposing fruits of Terminalia chebula, Oct.
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5. 2008, L.S. Yadav; AMH 9279, in vitro culture no. NFCCI
1494.
Additional cultures examined: NFCCI 2199 isolated
from partially degraded T. chebula fruits collected from
Mahabaleshwar, Maharashtra, India, August 2009, L.S.
Yadav; NFCCI 1496 (G. simplex), Mahabaleshwar, India,
on decomposing fruits of T. chebula, Oct. 2008, L.S.
Yadav.
Internal transcribed spacers (ITS) and 5.8S rRNA gene
sequence similarity confirmed the identity of the isolates
with [99% sequence homology with respective species.
For comparison, rDNA sequences (ITS regions including
5.8S) of Indian isolates were aligned with authentic
sequences available in the GenBank database including
those from ex-type strains of all known Gliocephalotri-
chum species using CLUSTAL W. The neighbor-joining
tree resulted in a monophyletic clade of Gliocephalotri-
chum with bootstrap value of 99% (Fig. 2). The Glio-
cephalotrichum clade formed three subclades: the first one
consists of G. bulbilium, G. cylindrosporum, and G. sim-
plex; the second subclade consists of G. microchlamydo-
sporum and G. ohiense; and the third subclade consists of
G. bacillisporum and G. longibrachium.
Strain NFCCI 1921 of G. longibrachium differed at four
positions (2 deletions, 1 insertion, and 1 transversion) from
the ex-type strain of G. longibrachium (MUCL 46693).
The ex-type strain of G. bulbilium (MUCL 3185) differed
from NFCCI 1494 in only one position in 478 nucleotides.
Aligning sequences from Indian strains with those of
authentic/ex-type strains, we recognized several species-
specific variable nucleotide positions within the ITS1 and
ITS2 region (TreeBase submission ID 12091).
The genus Gliocephalotrichum, established with the type
species G. bulbilium J.J. Ellis & Hesselt., contains seven
species described mostly from tropical or subtropical
countries. The geographic distribution of this genus is
Japan, Thailand, French Guiana, South Africa, Brazil,
Malaysia, India, and the United States (Decock et al. 2006;
Nishijima et al. 2002; Watanabe and Nakamura 2005;
Haung and Schmitt 1973). The species concept of this genus
is based on distinguishing characters, such as position of
sterile arms on conidiogenous penicillus; sterile arms sub-
tending to penicillus (G. bulbilium, G. microchlamydospo-
rum, G. ohiense, G. bacillisporum, and G. longibrachium),
and sterile arms arising at some distance below the peni-
cillus (G. simplex and G. cylindrosporum). Nicot (1967)
Fig. 1 Gliocephalotrichum longibrachium: habit showing conidiophores with sterile arms (a); chlamydospore (b); conidia (c). Gliocephalo-
trichum bulbilium: habit showing penicillus and sterile arms (d); chlamydospores (e); conidia (f). Bars a, d 40 lm; b 60 lm; c, d, f 20 lm
Mycoscience (2012) 53:391–395 393
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6. noticed some variability in location of the sterile arms,
especially in G. bulbilium. The isolate of G. bulbilium
obtained in the present study showed differences in the
characters from the type description of the species. They
differed in growth rate (80 mm in 3 days vs. 80 mm in
10 days), hyphal/sterile arms (numbers: 2–9 vs. 1–4 or
more; size: up to 550 vs. 100–250 lm), conidiophores
(185–575 vs. 160–340 lm), chlamydospores (35–112 vs.
50–90 lm), metulae, and conidia. Variation in the mor-
phology of G. longibrachium was also recorded when
compared with the original type description of G. longib-
rachium (Decock 2006). Conidiophores were longer
(140–225 lm) in the Indian isolates and conidia smaller
(5–8.5 lm) than in the French Guiana isolates (Decock
et al. 2006). The aggregated chlamydospore cells form
different shapes.
Soil and leaf litter (Wiley and Simmons 1971; Haung
and Schmitt 1973; Decock et al. 2006; Jalmi et al. 2011)
have been reported to be the habitats for species of Glio-
cephalotrichum. Arya (1994) reported Gliocephalotrichum
on decayed fruits. In our studies we observed Gliocepha-
lotrichum predominantly colonizing T. chebula fruits,
suggesting the possibility of selective invasion and devel-
opment on these fruits. Search for members of the genus on
other fallen fruits collected in the same locality proved
negative. It appears worthwhile to focus on mycobiota of
decomposing fruits in contact with litter to identify novel
and unique taxa. As such, our study on decomposing fruit
in which we obtained three Gliocephalotrichum species on
the same fruit indicates that fruit litter may harbor spe-
cialized fungal flora involved in decomposition.
Natural fungal resources are considered to contribute a
great deal to biotechnological applications (Hoffmeister
and Keller 2007). The biotechnology application potential
of Gliocephalotrichum has not been explored, except in
some reports indicating them as a source of melanin for use
in plastic laminates for optical applications (Gallas and
Eisner 2006) and in skin care formulations (Jalmi et al.
2011). It would be desirable to explore the biodiversity of
the genus from diverse habitats and explore them for
Fig. 2 Internal transcribed
spacers (ITS) and 5.8S rRNA
gene sequence-based
phylogenetic relationships of
Gliocephalotrichum bulbilium
(NFCCI 1494) and G.
longibrachium (NFCCI 1921)
with other strains of
Gliocephalotrichum. The
phylogenetic tree was drawn
using 610 nucleotides of ITS 1,
2, and 5.8S rRNA gene
sequences using the neighbor-
joining method in MEGA v.5
software. Bar represents the
distances calculated in MEGA;
values at nodes indicate the
bootstrap percentages.
Bootstrap values less than 50%
are not shown
394 Mycoscience (2012) 53:391–395
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7. valuable secondary metabolites and industrial enzymes
with novel applications. Work on standardization of vari-
ous fermentation parameters, such as pH, temperature,
aeration, and media composition for the production of
melanin by Gliocephalotrichum spp. described in this
article, is in progress, and preliminary results obtained are
promising.
Acknowledgments The authors thank two unknown reviewers for
their constructive criticism and editing of our manuscript and Dr.
M.C. Srinivasan for his critical suggestions in improving the manu-
script. The authors also thank the Director, MACS’Agharkar
Research Institute, Pune, for providing the facility and the Depart-
ment of Science and Technology (DST), Govt. of India, New Delhi,
for setting up the National Facility for Culture Collection of Fungi
(No. SP/SO/PS-55/2005) at ARI. The authors do not have any conflict
of interest.
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